Expression of Ca2+-dependent synaptotagmin isoforms in mouse and rat parotid acinar cells

Abstract

Synaptotagmin is a Ca2+ sensing protein, which triggers a fusion of synaptic vesicles in neuronal transmission. Little is known regarding the expression of Ca2+-dependent synaptotagmin isoforms and their contribution to the release of secretory vesicles in mouse and rat parotid acinar cells. We investigated a type of Ca2+-dependent synaptotagmin and Ca2+ signaling in both rat and mouse parotid acinar cells using RT-PCR, microfluorometry, and amylase assay. Mouse parotid acinar cells exhibited much more sensitive amylase release in response to muscarinic stimulation than did rat parotid acinar cells. However, transient [Ca2+]i increases and Ca2+ influx in response to muscarinic stimulation in both cells were identical, suggesting that the expression or activity of the Ca2+ sensing proteins is different. Seven Ca2+-dependent synaptotagmins, from 1 to 7, were expressed in the mouse parotid acinar cells. However, in the rat parotid acinar cells, only synaptotagmins 1, 3, 4 and 7 were expressed. These results indicate that the expression of Ca2+-dependent synaptotagmins may contribute to the release of secretory vesicles in parotid acinar cells.

Fig. 1

Concentration-response curve of muscarinic agonist, carbachol, on amylase release and carbachol-induced [Ca²⁺]_i increases in parotid gland acinar cells from rats and mice. A, Amylase releases were measured with stimulation of the muscarinic agonist, carbachol, in a concentration range of 0.1 µM to 100 µM in parotid acinar cells from rats and mice. Results are expressed as the mean ± S.E.M. of four experiments in each group. B, [Ca²⁺]_i in parotid acinar cells was measured with the relative ratio of fura2 fluorescence in response to each concentration of carbachol for 20 mins. The traces are representatives of 7 different experiments. C, analysis of carbachol-induced [Ca²⁺]_i initial peaks. D, analysis of carbachol-induced Ca²⁺ influx. Results are expressed as the mean ± S.E.M. of seven experiments in each group. ^*Significant difference between rat and mice parotid acinar cells (p<0.05).

Fig. 2

RT-PCR products encoding Ca²⁺-dependent synaptotagmins in parotid acinar cells and Western blotting of synaptotagmin 1 in exocrine glands. A, RT-PCR results of the Ca²⁺-dependent synaptotagmin family in brain microsome and parotid acinar cells from mice and rats. Lane numbers indicate synaptotagmin isoforms. B, size of PCR products encoding synaptotagmin isoforms and summary of results. Note that rat parotid acinar cells express synaptotagmin isoforms 1, 3, 4, and 7. Each result is the representative of five to eight different experiments. C, Western blotting of synaptotagmin 1 in brain, parotid, submandibular, and pancreatic acinar cells from mice and rats. The brain contains 40 µg and the parotid, submandibular, and pancreatic acinar cells contain 100 µg of proteins in each lane. Each result is the representative of four different experiments. -Ex; no extract.